Urine pcr assay and use thereof to diagnose and stage feline chronic kidney disease

Abstract

This invention is related, generally, to a novel assay for Feline Chronic Kidney Disease (CDK) and, more particularly, to detecting the presence of a target compound in a sample which may contain the target compound, amplification, and subsequent quantitation of the detector molecule to permit both the diagnosis and staging of feline CDK. The present assay is particularly useful for diagnosing early CDK, when clinical signs may not be present, to permit intervention and treatment. Another embodiment provides target compounds, such as antibodies, and methods using these compounds for the treatment of renal fibrosis associated with chronic kidney disease. In one embodiment, the present invention provides a pharmaceutical composition comprising a mRNA encoding a chimeric antigen receptor (CAR) antibody, and a transfer vehicle comprising a lipid nanoparticle (LNP), and methods of producing CAR-T cells that limit the progression of renal disease in mammal.

What is claimed is: 
     
         1 . A PCR diagnostic assay for differentially identifying early stage Chronic Kidney Disease (CDK) from late stage CDK in a subject comprising quantifying the amount of fibroblast activation protein (FAP) nucleic acid present in a sample from the subject. 
     
     
         2 . The PCR diagnostic assay of  claim 1 , wherein the subject is a companion animal selected from cats, dogs, or horses. 
     
     
         3 . The PCR diagnostic assay of  claim 2 , wherein the companion animal is a cat. 
     
     
         4 . The PCR diagnostic assay of  claim 1 , wherein the sample is urine. 
     
     
         5 . The PCR diagnostic assay of  claim 1  wherein the nucleic acid is mRNA. 
     
     
         6 . The PCR diagnostic assay of  claim 5  wherein the mRNA is isolated from endosomes in the urine. 
     
     
         7 . A PCR diagnostic assay for differentially identifying early stage Chronic Kidney Disease (CDK) from late stage CDK in a subject comprising:
 (a) obtaining a urine sample from a subject;   (b) isolating mRNA from an endosome in the urine;   (c) using the mRNA as a template to produce cDNA by reverse transcription;   (d) amplifying the cDNA by reacting the cDNA with primers to make an amplification product; and   (e) quantitating or detecting the amplification product.   
     
     
         8 . The PCR diagnostic assay of  claim 7 , wherein the subject is a companion animal selected from cats, dogs, or horses. 
     
     
         9 . The PCR diagnostic assay of  claim 8 , wherein the companion animal is a cat. 
     
     
         10 . The PCR diagnostic assay of  claim 7 , the primers comprise a forward primer having a nucleotide sequence as set forth in SEQ ID NO: 6 and a reverse primer having a nucleotide sequence as set forth in SEQ ID NO:7. 
     
     
         11 . The PCR diagnostic assay of  claim 10  further comprising a DNA polymerase; blocking oligonucleotides; buffers, and a detectable label. 
     
     
         12 . The PCR diagnostic assay of  claim 7 , wherein the amplification product is analyzed using a probe specific for FAP to determine if FAP is present. 
     
     
         13 . The PCR diagnostic assay of  claim 12  wherein the probe includes a fluorophore as a fluorescent label. 
     
     
         14 . The PCR diagnostic assay of  claim 13 , wherein the fluorophore is selected from CalO 560, CalRed 610, or FAM. 
     
     
         15 . A PCR diagnostic assay for differentially identifying early stage Chronic Kidney Disease (CDK) from late stage CDK in a subject comprising
 (a) extracting mRNA from the sample obtained from the subject;   (b) using a reverse transcriptase reaction to obtain a cDNA template;   (c) adding the cDNA, a positive control, and optionally a negative control into PCR tubes of a PCR reaction system respectively to obtain a corresponding sample reaction tube, positive reaction tube or negative reaction tube, wherein the PCR reaction system contains the primers for detecting FAP;   (d) performing a PCR reaction by placing the reaction tubes on a PCR instrument, setting parameters, and performing PCR reaction to produce an FAP amplification product;   (e) quantifying the FAP in the sample as compared to a control; wherein the presence of FAP amplification product indicates the presence of fibrosis.   
     
     
         16 . The PCR diagnostic assay of  claim 15 , wherein the subject is a companion animal selected from cats, dogs, or horses. 
     
     
         17 . The PCR diagnostic assay of  claim 16 , wherein the companion animal is a cat. 
     
     
         18 . The PCR diagnostic assay of  claim 15 , wherein the sample is urine.